Pharmaceutical Microbiology

5PY017 Pharmaceutical Microbiology

 

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Evaluation of antimicrobial effectiveness of disinfectants

 

There are a number of methods available to assess the effectiveness of antimicrobial products such as disinfectants and antiseptics.  The European and British Standard methods consist of a range of methods to cover medical, veterinary and food/industrial areas.

This practical is based on BS EN1276:1997 ‘Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas – test method and requirements ’.  The disinfectant benzalkonium chloride will be tested at three concentrations against Gram positive (Staphylococcus aureus and Enterococcus hirae) and Gram negative (Pseudomonas aeruginosa and Escherichia coli) bacteria in conditions to simulate usage (presence of organic load and hard water).  To pass the test a disinfectant must reduce the count of the four organisms by five log orders within the five minute contact time.

 

Working individually each students will test one organism and one disinfectant concentration. Class data will be collected to provide a full set of data.

Aseptic microbiological techniques must be used throughout the practical.

 

Method

 

Enumeration of the concentration of the bacterial suspensions.

 

  1. Record which bacterial suspension and disinfectant concentration are to be tested:

Bacterium……………………..

Concentration of………………

 

  1. Label duplicate Petri dishes with student name, name of the organism, and 10-6,10-7

 

  1. Take the bacterial suspension labelled and perform a decimal dilution by removing 0.5ml of the suspension and adding to 4.5ml Ringers to give a 10-1 dilution.  Mix and then continue the dilution series to 10-7 and pour plate in duplicate 1ml aliquots from the 10-6 and 10-7 dilutions.

 

Keep the 10-5 dilution and label ‘D’ (Diluted) as this will be used to inoculate controls in the test.

 

 

 

Controls

These have been prepared for you and the results will be provided.

 

A and B are controls to ensure that the medium and neutraliser do not affect the microorganisms.  Control C is to check that the neutraliser is effective in inactivating the disinfectant at the end of the contact time.

 

Control A contained 1ml bovine serum albumin, 1 ml of bacterial suspension (10-5) and 8ml sterile distilled water and then vortexed.  After 5 min1.0 ml aliquots were removed and for pour plated in duplicate.

 

Control B contained 8ml neutraliser, 1ml sterile distilled water and 1ml of bacterial suspension (10-5) and vortexed.  After 5 min 1.0 ml aliquots wereremoved and pour plating in duplicate

 

Control C contained 1ml bovine serum albumin, 1ml sterile distilled water and 8ml disinfectant (0.5% BAC) and vortex. After 5 min, 1 ml was transferred to a tube containing 8ml neutraliser and vortexed. After 5 min, add 1ml bacterial suspension (10-5) was added. After a further 5 min, 1 ml aliquots were pour plated in duplicate.

 

Test

 

  1. Label duplicate petri dishes with your name, organism and disinfectant concentration.
  2. To a test tube add 1ml bovine serum albumin and 1ml bacterial NEAT suspension (coloured lid) and vortex.  Add 8ml disinfectant, vortex and after exactly 5 min, transfer 1ml to a second tube containing 8 ml neutraliser and 1 ml sterile distilled water tube and vortex. After 5 min, remove duplicate 1.0 ml for pour plating.
  3. Incubate all plates at 37oC for 24 hr.

 

 

Summary of the overall test

 

 

 

 

 

Individual Results

 

Count the cfu/plate and record in the tables below.

 

Bacterial Culture…………………………..

 

Table 1 Calculation of concentration of bacterial suspension N

  10-6 10-7
Duplicate 1    
Duplicate 2    
Mean cfu ml-1    
Mean cfu ml-1 in undiluted* N  

For results that are too numerous too count, record as >300

*For calculation of cfu ml-1, use the dilution which gave 30-300 colonies

 

Table 2 Control Results – these will be provided

 

E.coli

  A B C
Duplicate 1      
Duplicate 2      
Mean cfu ml-1      

 

S.aureus

  A B C
Duplicate 1      
Duplicate 2      
Mean cfu ml-1      

 

 

 

P.aeruginosa

  A B C
Duplicate 1      
Duplicate 2      
Mean cfu ml-1      

 

E.hirae

  A B C
Duplicate 1      
Duplicate 2      
Mean cfu ml-1      

 

 

 

Table 3 Test – note concentration tested

  …………%
Duplicate 1  
Duplicate 2  
Mean cfu ml-1  
Mean x10 cfu ml-1 in test mixture T  

Calculation of Reduction Factor (RF) for each concentration of disinfectant

 

RF=  NX10-1

             T

Where                        N is the number of cfu ml-1 in the bacterial suspension

(from the shaded box in Table 1 above)

T is the number of cfu ml-1 in the test mixture for (from the shaded boxes in Table 3 above)

            

To pass the disinfectant test, the in-use concentration (0.1%) must give a reduction factor of>105 for all four organisms within the 5 minute contact time.

                                                                                                                                                                                                                       

 

 

 

 

 

 

 

 

 

 

 

 

 

Class results

Collect class data of the reduction factors for all four organisms and three concentrations of the disinfectant benzalkonium chloride

 

  Reduction Factor (RF)
   

0.005      % BAC

 

0.1       % BAC

 

0.5

% BAC

S.aureus

 

 

 

 

 

 

     
E.coli

 

 

 

 

 

 

     
P.aeruginosa

 

 

 

 

 

 

     
E.hirae

 

 

 

 

 

 

     

 

 

 

 

Practical write-up

This should be written as a full write-up as follows

Abstract– a concise and detailed summary of the findings

 

Introduction – provide a background to disinfectant testing, the organisms included and BAC

 

Materials and methods– do not write out the methods, refer to the practical schedule only and note any changes

 

Results and discussion

 

For your own results comment on the controls, the results obtained for the organism tested and validity of the test.

 

For the Class results – discuss the results obtained with respect to repeatability, concentrations tested, the susceptibilities of the organism used and the overall test outcome.

 

Referencing

You should be referring to and citing literature sources in your introduction and discussion sections.  Please note advice on referencing.

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